INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

THE AGING PROCESS has a marked impact on structural and functional characteristics of human skeletal muscle that may compromise contractile function. One negative effect of aging is skeletal muscle atrophy, which could contribute to the reduction in muscular strength reported in aged individuals (1, 8, 10, 15, 22). The effect of aging on the energy-generating systems in skeletal muscle is, however, less clear. Several studies have demonstrated little or no change in muscle glycolytic enzyme activity with aging (1, 12, 15, 22). Findings concerning muscle oxidative capacity are more controversial because some studies report no alteration (1, 2, 12, 15, 16, 22, 27), whereas others report a decrease (9, 10, 28), in oxidative enzyme activities. In addition, some data indicate a decrease in the relative percentage of the glycolytic, type II fibers (20, 22, 23, 27), whereas others indicate no change (1, 2, 12, 21, 24, 25), with aging. Such findings are relevant because impairments in energy production may contribute to aging-related conditions such as increased risk for falls, declining mobility, and other functional disabilities.

The lateral gastrocnemius and the vastus lateralis are the leg muscles commonly sampled in human aging studies; both are also involved in many daily activities (4). We recently reported that fiber composition and oxidative enzyme activity differed between these two muscle groups within the same young individuals (17). The exposure to contractile activity throughout the life span may also differ markedly; electromyographic studies suggest that the gastrocnemius is more tonically controlled, whereas the vastus is recruited primarily during locomotion (4). Part of the controversy surrounding the impact of aging on skeletal muscle in relation to oxidative capacity may thus be a result of combining data from functionally distinct muscle groups. In the present study, we attempted to clarify this issue by comparing the effects of aging on enzymatic (citrate synthase activity) and histochemical (fiber type) characteristics of the vastus lateralis and the gastrocnemius in humans.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Subjects. Sedentary Caucasian men who were 18-80 yr of age were studied. Inclusion criteria for all subjects included no regular exercise in the previous 2 yr and no evidence of cardiovascular disease or diabetes (fasting plasma glucose concentration >7.7 mmol/l on 2 occasions). On the basis of verbal questioning and a medical history, we determined that the aged subjects had led a largely sedentary lifestyle with no prolonged periods (>3 yr) of a regular exercise program; this was supported by the maximal oxygen consumption (O_{2 max}), body composition, and muscle (oxidative enzyme activity, fiber type) data (see RESULTS). All subjects were nonsmokers; a total of 55 men were studied. The experimental protocol was approved by the University Policy and Review Committee on Human Research.

Cardiorespiratory fitness. Fitness was determined by the O_{2 max} elicited during an incremental treadmill test. The Bruce protocol (6) was utilized in subjects up to age 40 yr. The Balke protocol (3) was used in subjects >40 yr; a modified Balke protocol, which utilized a slower speed (67.1 m/min), was used in the oldest subjects (60 yr of age). Different protocols were used so that exhaustion was reached within ~8-15 min, as recommended for a valid O_{2 max} test (7, 19). Criteria for data inclusion were a respiratory exchange ratio >1.0, voluntary fatigue, and attainment of at least 85% of maximal heart rate; similar criteria have been used in other studies (7, 19). Expired gases were monitored continously (Beckman Horizon MMC, SensorMedics, Anaheim, CA) for determination of oxygen uptake. Twelve-lead electrocardiogram (ECG) tracings were aquired every minute, and a resting ECG was obtained before treadmill testing.

Adiposity. Body density was determined by hydrostatic weighing, and body fat percent, fat mass, and fat-free mass were computed as described previously (17). Height was measured to the nearest 0.1 cm, and mass was measured to the nearest 0.1 kg.

Muscle characteristics. A sample from the lateral gastrocnemius and vastus lateralis was obtained with the percutaneous needle-biopsy technique; a portion was immediately frozen in liquid nitrogen for subsequent analysis of citrate synthase activity at 22°C (30). Part of the biopsy was mounted in a trigacanth gum-OCT (Miles, Elkhart, IN) mixture and frozen in isopentane cooled over liquid nitrogen. Mounted muscle was subsequently sectioned (10 µm) at 20°C and stained at pH 10.3 and 4.54 for determination of fiber type (5). Fiber type [vastus lateralis, 331 ± 23 (SE) fibers counted; gastrocnemius, 326 ± 21 fibers counted] was determined from the vastus lateralis of 46 subjects and from the gastrocnemius of 45 subjects; citrate synthase activity from the gastrocnemius was determined in 52 subjects and from the vastus lateralis of 53 subjects.

Statistical analysis. Univariate and multivariate linear regression analysis determined relationships between variables of interest in the population (age range 18-80 yr) studied. To determine differences between the maximal age ranges of the population (young vs. aged), data were also compared with a factorial analysis of variance in subgroups of the youngest (30 yr) vs. oldest (60 yr) individuals. Statistical significance was accepted at the P < 0.05 level.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Subject characteristics. Descriptive data for the 55 subjects are presented in Table 1. Body fat percentage (r = 0.46, P < 0.001) was positively associated with age in this population. Descriptive characteristics of the young (30 yr, n = 12) vs. aged (60 yr, n = 10) subgroups, respectively, were as follows: age, 23.7 ± 0.8 vs. 70.8 ± 2.2 yr; O_{2 max} (ml · kg¹ · min¹), 41.6 ± 1.2 vs. 23.6 ± 1.7; O_{2 max}, 3.5 ± 0.1 vs. 2.0 ± 0.2 l/min, and body fat 22.1 ± 1.3 vs. 28.0 ± 1.0%. All of these variables were significantly different between the young and aged subgroups (P < 0.001). Fat mass was significantly lower in the young vs. aged individuals (18.9 ± 1.7 vs. 23.8 ± 1.5 kg, respectively; P < 0.05); however, fat-free mass (65.3 ± 1.8 vs. 61.0 ± 2.1 kg) and body mass index (26.1 ± 0.9 vs. 27.9 ± 1.0 kg/m²) did not significantly differ between the young vs. aged subgroups, respectively.

O_{2 max}. Absolute O_{2 max} for the 55 men ranged from 1.5 to 4.9 l/min. Relative O_{2 max} ranged from 14.9 to 51.1 ml · kg¹ · min¹. The relationships between O_{2 max} and age in this population of men are presented in Fig. 1. There was a significant (P  0.001) negative relationship between age and O_{2 max} expressed in either relative (r = 0.85) or absolute (r = 0.80) terms. The rate of decline in O_{2 max} (0.40 ml · kg¹ · min¹ · yr¹ and 0.033 l · min¹ · yr¹) agrees with other data (7, 19, 31).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Relationships of age with maximal oxygen consumption (O2 max). Decline in relative O2 max (A) was 0.40 ml · kg1 · min1 · yr1; rate of decline in absolute O2 max (B) was 0.033 l/min. n = 55 Men.

Muscle characteristics. Mean citrate synthase activity in the gastrocnemius was 18.3 ± 0.7 vs. 13.1 ± 0.4 µmol · g wet wt¹ · min¹ in the vastus lateralis (P < 0.001) in the population studied. As presented in Fig. 2, citrate synthase activity in the gastrocnemius was negatively related to age (r = 0.32, P < 0.05) in this population of men; there was no relationship between citrate synthase activity in the vastus lateralis and age (r = 0.13, P = 0.37). A similar pattern was evident when the subgroups of the youngest and oldest men were compared (Fig. 3); citrate synthase activity was significantly lower (P < 0.05) in the older men only in the gastrocnemius, and the value was lower by ~24%. In the younger men, citrate synthase activity was significantly (P < 0.05) lower in the vastus lateralis compared with the gastrocnemius. As presented in Fig. 4, in this population relative O_{2 max} on the treadmill was significantly related to citrate synthase activity in the gastrocnemius (r = 0.43, P < 0.01) but not in the vastus lateralis (r = 0.09, P = 0.54); there were similar relationships (data not shown) between absolute O_{2 max} and citrate synthase activity in the gastrocnmemius (r = 0.35, P < 0.05) and the vastus lateralis (r = 0.02, P = 0.88).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Relationships of citrate synthase activity (µmol · g wet wt1 · min1) in gastrocnemius (A; n = 52 men) and vastus lateralis (B; n = 53 men) with age (yr).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Comparison of citrate synthase activity (µmol · g wet wt1 · min1) in gastrocnemius and vastus lateralis in young (n = 12) vs. old (n = 10) men. * P < 0.05 vs. gastrocnemius muscle from young men. § P <0.05 vs. vastus lateralis muscle from young men.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Relationships between citrate synthase activity (µmol · g wet wt1 · min1) in gastrocnemius (A; n = 52 men) and vastus lateralis (B; n = 53 men) with O2 max.

Stepwise regression. Results of stepwise regression analysis in the men studied are presented in Table 2. Variables entered were fat-free mass, percent fat, fat mass, muscle fiber composition, and citrate synthase activity of both muscle groups; only subjects with all of these data (n = 35) were entered into the regression analysis. Regression analysis indicated that percent fat accounted for 53% of the variance in relative O_{2 max}, followed by citrate synthase activity of the gastrocnemius (7%) and percentage of type IIb of the vastus lateralis (7%). For predicting absolute O_{2 max}, regression analysis indicated that fat-free mass accounted for 34% of the variance followed by percent fat (19%), citrate synthase activity in the gastrocnemius (7%), and percentage of type IIb of the vastus lateralis (7%).

View this table: [in this window] [in a new window]   Table 2.   Stepwise regression analysis of body composition, muscle citrate synthase activity, and muscle fiber composition with O2 max

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The impact of aging on enzymes that reflect respiratory capacity in human skeletal muscle is not clear. The data from the present study suggest that, in humans, distinct leg muscles may respond differently to the aging process. More specifically, muscle oxidative capacity appeared to decrease in the gastrocnemius but not in the vastus lateralis with advancing age. This conclusion was deduced from our findings of 1) a negative relationship between age and citrate synthase activity in the gastrocnemius but not in the vastus lateralis in a population of men (Fig. 2) and 2) a decline in citrate synthase activity (24%) in the lateral gastrocnemius but not in the vastus lateralis when a subgroup of young (30 yr) vs. aged (60 yr) men was directly compared (Fig. 3).

This finding is not in agreement with the data of Rooyackers et al. (28), who reported a negative relationship between citrate synthase activity in the vastus lateralis and age in a population of men and women. Our data do agree with other work (1, 2, 12, 15, 16, 22, 27) that found no change in oxidative enzyme activities in the vastus lateralis with aging. Studies that have sampled the gastrocnemius have reported a similar decline (25%) in oxidative enzyme activity (9, 10). No previous work, however, has directly compared the biochemical characteristics of these two muscle groups in sedentary young vs. aged individuals. Grimby et al. (16) reported a decrease in muscle fiber area with aging in the vastus lateralis but not in the biceps brachii; muscle citrate synthase activity was not, however, altered in either muscle group. These data and the present findings suggest that citrate synthase activity and oxidative capacity either decreases or is not altered with the aging process in human skeletal muscle; this depends, however, on the muscle group studied.

The explanation for a reduction in citrate synthase activity in the gastrocnemius but not the vastus lateralis with aging is not clearly evident. Some data (4) suggest that the gastrocnemius is more tonically stimulated than is the vastus lateralis. It is possible that tonically active muscle groups are more susceptable to the impact of the aging process. Self-reported physical activity is thought to decrease with aging (7, 19, 31). Although all subjects were determined to be sedentary on the basis of questioning and their O_{2 max}, body composition, and muscle characteristics (fiber type, oxidative enzyme activity), it is possible that lower levels of spontaneous physical activity in the aged group contributed to the reduction in muscle oxidative capacity in the gastrocnemius. It is not evident, however, why the gastrocnemius would be more dramatically affected by a change in activity levels with aging than would the vastus lateralis. There is also evidence for a change in the neurogenic stimulus to skeletal muscle with aging that can lead to structural changes (1, 2, 12, 15, 16, 24, 25). Certain muscle groups, such as the gastrocnemius, could respond more profoundly to a change in neurogenic stimuli with aging and exhibit an accompanying reduction in mitochondrial content. The explanation for the different responses of specific leg muscles to aging observed, however, remains to be more clearly elucidated with further study.

The contractile and energy-producing charactistics of the gastrocnemius and vastus lateralis may influence functional capacity with aging (8, 14). Findings in the gastrocnemius are important because a reduction in plantar flexor strength and endurance in the elderly is related to an increased incidence of falls (29). In young individuals, it has been hypothesized that the pattern of tension development and energy production is closely synchronized between the vastus lateralis and gastrocnemius during activities such as locomotion (14). With aging, the reduction in oxidative capacity in the gastrocnemius noted in the present study may disrupt this coordination and contribute to a functional impairment. Such interactions indicate it is vital to explore potential mechanisms in skeletal muscle that may contribute to limitiations in physical ability with aging.

In other work comparing the human vastus lateralis and gastrocnemius, Green et al. (14) reported increased lactate dehydrogenase activity in the vastus lateralis but no differences in other enzymes indicative of energy production (succinate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, total phosphorylase) or fiber type in young (20-24 yr), athletic individuals. In sedentary, young individuals there are trends for the gastrocnemius to be more oxidative than the vastus lateralis in terms of fiber type and enzyme activities, although the number of subjects studied has been small (11, 13). Because our young subjects were sedentary, we are in agreement with these latter findings (Fig. 3). These data suggest that fitness level and age are important factors that influence the characteristics of these two muscle groups within an individual; however, more study needs to be done to conclusively define these relationships.

Fiber type was studied because it can be indicative of the metabolic properties of muscle. Again, the literature is controversial as in the vastus lateralis an increase in the relative percentage of type I fibers (22, 27) or no change (1, 2, 12, 21, 24, 25) in fiber type has been reported with aging. The data from the present study support the premise that fiber type is not altered with aging; therefore, any alterations in functional capacity cannot be attributed to a change in muscle fiber composition. The present study did not, however, investigate the degree of muscle atrophy that occurs with advancing age; such atrophy would alter exercise tolerance and functional capacity (1, 2, 9, 10, 12, 21-25).

The decline in O_{2 max} with aging is well documented and has been attributed to many factors (7, 19, 31). The decrease in citrate synthase activity in the gastrocnemius observed suggests that a reduction in skeletal muscle oxidative enzyme activity may parallel the decline in O_{2 max} with the aging process. Citrate synthase activity was measured because it is found in direct proportion to muscle mitochondrial content (18) and correlates highly with in vivo oxidative metabolism in humans (9, 26). These data do not, however, provide solid evidence for a cause-and-effect relationship. It has been proposed that many of the detrimental effects of aging, including the decline in O_{2 max}, increased body fat, and reduction in muscle functional capacity, are secondary to a reduction in physical activity (7, 19, 31). The decline in O_{2 max} and citrate synthase activity in skeletal muscle and the increased body fat in the present study could thus reflect concommitant changes with a reduction in physical activity with the aging process.

In conclusion, not all muscles in humans appear to be affected similarly by the aging process. In the present study there was a decrease of citrate synthase activity in the gastrocnemius but not in the vastus lateralis with advancing age. These data suggest that the decline in mitochondrial capacity in human skeletal muscle is not uniform and may be muscle-group specific.